Fluorescent and colored compounds have been used in the fields of biological research and medicine to detect the presence, absence, state, quantity, and composition of biomolecules. Assays using fluorescent and colored compounds may be performed in vitro, in situ, or in vivo. Examples of commonly used in vitro assays for detection of DNA and RNA are real-time and end-point polymerase chain reaction (PCR), DNA sequencing, and DNA microarray technologies.
Recently, there has been an increased amount of literature published on detection methods for multiple analytes, most of which involves genetic analysis and some relates to protein detection. See, e.g., U.S. Pat. No. 6,890,741.
In a typical nucleic acid detection method used for diagnostic and molecular biology research, multiple gene probes complementary with a gene of interest are labeled with small molecules that can be detected by spectroscopic, electrochemical, biochemical or immunochemical means. PCR is generally incorporated for the amplification of targeted gene sequences. To achieve detection of multiple analytes, fluorescence-based technologies have been used often due to fluorescence dyes readily available. For example, primers have been labeled with different fluorescence dyes and the changes in fluorescence were monitored upon hybridization to their complements (e.g., WO 2002/057479). In other cases, the multiplex detection was achieved by using intercalating dyes as labels in DNA restriction fragment analysis and capillary electrophoresis with frequency-domain fluorescence lifetime detection method (McIntosh, et al., Electrophoresis, 2002, 23, 1473-1479). Since these methods use pre-labeled fluorescence dyes, the detection sensitivity relies largely on the separation of target bound and unbound fluorescence labeled probes. Though solid phase immobilization of the target gene (fluorescence in situ hybridization, for example) can improve the separation efficiency by simply washing away the unbound fluorescence labeled probes, this introduces an extra process. However, the potential background still can be high, and the procedure can be laborious. To address this problem, a non-fluorescence label moiety can be attached to the probes so that the fluorescence signal only occurs after the hybridization event. Recently, the development of DNA-programmed chemistry has provided a novel approach for generation of fluorescence dye in situ. See, e.g., Li, X.; Liu, D. R. Angew. Chem. Int. Ed. 2004, 43, 4848-4870; U.S. Pat. No. 7,070,928.
Polymethine dye has been widely used as laser dyes, photographic sensitizers and fluorescence probes due to its superior fluorescence and photochemical properties. However, polymethine dyes are generally synthesized by acid/base catalyzed condensation under anhydrous conditions which is not comparable to the nucleic acid-templated chemistry (Jedrzejewska, et al. Dyes and Pigments 2003, 58, 47-58). Recently, the literature has reported an improved aldol condensation in water using Lewis-acid (Kobayashi, et al., J. Am. Chem. Soc. 1998, 120, 8287-8288) and enamine-based organocatalyst (Mase, et al. J. Am. Chem. Soc. 2006, 128, 734-735). The quaternary salt of polymethine precursor (active hydrogen component) used for condensing with aldehyde, however, is different substantially from the precursor (alpha carbon of aldehyde) in a conventional aldol condensation.
Thus, there exists a need for new fluorescent and colorimetric technologies that address many of the shortcomings inherent in the above-mentioned biodetection methods. For example, there is a need for methods of polymethine dye synthesis from non-detectable precursors by nucleic acid-templated chemistry and adaptation of such chemistry to biodetection.